Calmangafodipir, a new chemical entity, and other mixed metal complexes, methods of preparation, compositions, and methods of treatment

ABSTRACT

Methods for treatment of a pathological condition caused by oxidative stress in a patient comprise administering to the patient a mixed metal complex of a compound of Formula I, or a salt thereof, in an amount effective to reduce the oxidative stress. The mixed metals comprise calcium and manganese in a molar ratio of calcium to manganese in the range of 1-10: 
                         
wherein X, R 1 , R 2 , R 3 , and R 4  are as defined herein.

RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 14/369,153 filed Jun. 26, 2014, which is a 371 ofPCT/IB2012/056959 filed Dec. 4, 2012, which claims priority to U.S.Provisional Applications Nos. 61/583,377 filed Jan. 5, 2012, 61/656,178filed Jun. 6, 2012, 61/668,679 filed Jul. 6, 2012, and 61/721,575 filedNov. 2, 2012.

FIELD OF THE INVENTION

The present invention is directed to a mixed metal complex of adipyridoxyl compound, for example,N,N′-bis-(pyridoxal-5-phosphate)-ethylenediamine-N,N′-diacetic acid(DPDP or fodipir) or other compounds of Formula I (hereafter PyridoxyLEthylDiamine derivatives or PLED-derivatives), wherein the mixed metalscomprise a Group III-XII transition metal and a Group II metal. Inspecific embodiments, the mixed metal complex is a calcium and manganesecomplex. The present invention is also directed to compositionscontaining such a mixed metal complex, methods for preparing such amixed metal complex, for example, in a single step crystallization, andtreatment methods employing such a mixed metal complex. Such treatmentmethods include methods conventionally employing manganese-DPDPcomplexes for therapeutic effect. In a specific embodiment, thecompositions may be used in the treatment of pathological conditionscaused by the presence of oxygen-derived free radicals in the body,i.e., oxidative stress. The mixed metal complexes, and particularly themixed calcium-manganese complex calmangafodipir described herein,constitute new chemical entities.

BACKGROUND OF THE INVENTION

Oxidative stress begins with the generation of reactive oxygen species(ROS) and reactive nitrogen species (RNS) as a part of normal cellularfunction. There are multiple cellular sources of ROS generation but themost significant ones are the mitochondria electron transport complexesI and III, P450 enzymes within the endoplasmic reticulum, and membranebound NADPH oxidase. ROS production by each of these sources can bestimulated by cytokines, inflammation, viral proteins, and othermechanisms, like chemotherapy drugs, ischemia-reperfusion, and iron andcopper overload. Importantly, these processes initially generate thefree-radical superoxide (.O₂ ⁻) which is sequentially reduced to formhydrogen peroxide, hydroxyl radical and, ultimately, water. Underconditions of high oxidative stress and consequently high production ofsuperoxide, these reactive intermediates, however, readily interact withother molecules to form secondary harmful ROS, such as lipidperoxidation products and peroxynitrite (Singal et al., Liver Int. 2011;31:1432-1448). This indicates the importance of keeping the cellularamount of superoxide under tight control. Under normal conditions thisis achieved by superoxide dismutases (SODs). Although SODs have thefastest reaction rate of known enzymes, under conditions of highoxidative stress, these enzymes may be outcompeted and even irreversibleirreversibly inactivated by ROS and RNS. This in turn, opens up fortherapeutic use of low molecular drugs that mimic the SOD enzymes, i.e.,the so-called SOD mimetics, to combat pathological oxidative stress.

Short-lived but highly reactive oxygen-derived free radicals have longbeen known to participate in pathological tissue damage, especiallyduring treatment with cytotoxics/cytostatics and radiotherapy in cancerpatients (Towart et al., Arch Pharmacol 1998; 358 (Suppl 2):R626,Laurent et al., Cancer Res 2005; 65:948-956, Karlsson et al., Cancer Res2006; 66:598, Alexandre et al., J Natl Cancer Inst 2006; 98:236-244,Doroshow, J Natl Cancer Inst 2006; 98:223-225, Citrin et al, Oncologist,2010; 15:360-371, Kurz et al., Transl Oncol 2012; 5:252-259),acetaminophen-induced liver failure (Bedda et al., J Hepatol 2003;39:765-772; Karlsson, J Hepatol 2004; 40:872-873), in ischemic heartdisease (Cuzzocrea et al., Pharmacol Rev 2001; 53:135-159) and invarious neurodegenerative diseases, including Alzheimer's disease,amyotrophic lateral sclerosis (ALS), Parkinson's disease, and multiplesclerosis (Knight, Ann Clin Lab Sci. 1997; 27:11-25). Overproduction ofoxygen-derived free radicals is also implicated in pathologicalconditions of iron overload (Rachmilewitz et al., Ann N Y Acad Sci.2005; 1054:118-23), for example, in thalassemia, sickle cell anemia andtransfusional hemosiderosis. Oxygen-derived free radicals are alsoimplicated in hepatitis-induced liver cirrhosis (Farrell et al., AnatRec 2008; 291:684-692) and in noise-induced hearing loss (Wong et al.,Hear Res 2010; 260:81-88).

The use of dipyridoxyl based chelating agents and their metal chelatesand certain manganese-containing compounds, in particular manganesechelates, in medicine is known. See EP 0910360, U.S. Pat. No. 6,147,094,EP 0936915, U.S. Pat. No. 6,258,828, EP 1054670, U.S. Pat. No.6,310,051, EP 1060174, and U.S. Pat. No. 6,391,895, for example, whichdisclose that certain chelating agents, in particular dipyridoxylchelating agents, and their metal chelates, are effective in treating orpreventing anthracycline-induced cardiotoxicity, radiation-inducedtoxicity, ischemia-reperfusion-induced injuries, and paracetamol(acetaminophen) induced liver failure, or from a more general point ofview, every pathological condition caused by the presence ofoxygen-derived free radicals, i.e., oxidative stress, in humans andanimals. Furthermore, the dipyridoxyl compound mangafodipir (MnDPDP) hasin addition and surprisingly been found to possess cytotoxic effectsagainst cancer cells (EP 16944338). However, as described in WO2009/078794 A1 and in Kurz et al., 2012, this is an inherent property offodipir (DPDP) alone or its dephosphorylated counterparts, DPMP andPLED, and not of the metal complex MnDPDP or its dephosphorylatedcounterparts, MnDPMP and MnPLED.

One of the MnPLED-derivatives, namely manganeseN,N′-bis-(pyridoxal-5-phosphate)-ethylenediamine-N,N′-diacetic acid(Manganese DiPyridoxyl DiPhosphate; MnDPDP), also known as mangafodipir,is approved for use as a diagnostic MRI contrast agent in humans.Interestingly, mangafodipir has also been shown to protect mice againstserious side effects of several cytotoxic/cytostatic drugs (doxorubicin,oxaliplatin, 5-fluorouracil and paclitaxel), without interferingnegatively with the anticancer effects of these drugs (Towart et al.,1998, Laurent et al., 2005, Karlsson et al., 2006, Alexandre et al.,2006, Doroshow, 2006, Kurz et al., 2012). Mangafodipir has been testedin one colon cancer patient going through palliative treatment with acombination of folinate, 5-fluorouracil and oxaliplatin (Yri et al.,Acta Oncol. 2009; 48:633-635). The preclinical data and the results fromthis single patient were so promising that clinical testing in cancerpatients has started. When it comes to the most troublesome side effectof oxaliplatin, namely oxaliplatin-induced sensory neurotoxicity, nopreclinical data exist, to the best of our knowledge, showing protectiveeffects of mangafodipir (Karlsson et al., Transl Oncol. 2012; 5:32-38).Yri et al., 2009, described that the patient received 15 full-doses of“Nordic FLOX”. In 14 of the cycles, the patient received pretreatmentwith mangafodipir. The patient received an accumulated dose of 1275mg/m² oxaliplatin, which is a dose likely to give neurotoxic symptoms.No neurotoxic symptoms were detected, except during the fifth cycle whenmangafodipir was deliberately left out and the patient experiencedperipheral sensory neuropathy. This suggests that mangafodipir mayprotect against peripheral neurotoxicity. After five cycles, theperformance status for the patient was drastically improved, and thedemand for analgesics was significantly reduced. Neutropenia did notoccur during any of the chemotherapy cycles.

A first feasibility study (MANFOL I) has been completed and positiveresults, including myeloprotective effects, have been reported to theSwedish Medical Agency and have been published (Karlsson et al., 2012).

Mangafodipir has also been described to protect mice againstacetaminophen-induced acute liver failure in mice (ALF) (Bedda et al.,2003; Karlsson, 2004). ALF is characterized by massive hepatocyte celldeath, a condition caused by glutathione depletion, oxygen-derived freeradicals and mitochondrial damage.

Mangafodipir is a pro-drug in the sense that it probably has to bemetabolized into N,N′-dipyridoxyl ethylenediamine-N,N′-diacetic acid(MnPLED) before it can exert cytoprotective effects during in vivoconditions (e.g., see Karlsson et al., Acta Radiol 2001; 42:540-547;Kurz et al., 2012). Manganese is an essential as well as potentiallyneurotoxic metal. It has been known for many years that under conditionsof chronic exposure to high levels of manganese, a syndrome ofextrapyramidal dysfunction similar to Parkinson's syndrome, althoughclinically a different disease entity, frequently occurs (seeScheuhammer & Cherian, Arch Environm Contam Toxicol 1982; 11:515-520).When a diagnostic MR imaging dose of mangafodipir is intravenouslyinjected into humans, about 80% of the administered manganese isreleased (Toft et al., Acta Radiol 1997; 38:677-689). Release ofparamagnetic manganese is in fact a prerequisite for the diagnostic MRimaging properties of mangafodipir (Wendland, NMR Biomed 2004;17:581-594). Elizondo et al., 1991 (Radiology 1991; 178:73-78) statedthat the fodipir moiety binds to the pyridoxyl 5′ phosphate receptor onhepatocytes and ensures a high intracellular concentration ofmangafodipir in the liver. This hypothesis was recently also suggestedin a paper by Coriat et al., (PLoS One 2011; 6:1-6, e27005). This is anice hypothesis but unfortunately an unproven and a very unlikely one,which fell out of fashion shortly after it had been presented. Whenmangafodipir is injected intravenously (i.v.) about 80% of the metalcomplex falls apart (Toft et al., Radiol 1997), and at every equimolarMn dose, MnCl₂ has an equal or better liver MR imaging contrast efficacythan mangafodipir (Southon et al., Acta Radiol 1997). Furthermore, afterinjection of mangafodipir almost all fodipir is recovered in the urine(the major part of it as PLED), whereas most manganese is recovered inthe feces (Hustvedt et al., Acta Radiol 1997; 38:690-699). On the otherhand, the therapeutic effects of mangafodipir (MnDPDP) and itsdephosphorylated counterparts MnDPMP(N,N′-dipyridoxylethylenediamine-N,N′-diacetate-5-phosphate) and MnPLEDdepend on the intact metal complex (Brurok et al., Biochem Biophys ResCommun. 1999; 254:768-721, Karlsson et al 2001; 42:540-547).

PLED-derivatives mimic the mitochondrial enzyme manganese superoxidedismutase (MnSOD) (Brurok et al., 1999). MnSOD protects the mammaliancell from the superoxide radical, a byproduct from oxygen metabolism,which is produced in fairly high amounts during normal aerobicconditions; no mammalians survive without a functional MnSOD. MnSOD hasthe fastest turnover number (reaction rate with its substrate) of anyknown enzyme (>10⁹ M⁻¹ s⁻¹) (Fridovich, J Exp Biol. 1998;201:1203-1209). Low molecular weight MnSOD mimetics may have turnoverrates close to that of native MnSOD (Cuzzocrea et al., 2001).Interestingly, physiological buffers containing transition metals likemanganese may have similar high turnover numbers (Culotta et al.,Biochim Biophys Acta. 2006; 1763:747-758). However, the importance ofnative SOD enzymes is consistent with a selection process favoringorganisms that elaborate a means of localizing transition metal catalystfor superoxide dismutation to parts of the cell where there is a highneed for such dismutation, e.g., mitochondria. Furthermore, results frommyocardial ischemia-reperfusion in anaesthetized pigs inevitably showthat the intact MnPLED, but not manganese per se, protects againstoxidative stress, seen as reduction in infarct size (Karlsson et al.,2001). Effective inactivation of superoxide is essential in preventinggeneration of very devastating hydroxyl radicals and peroxynitrite(Cuzzocrea et al., 2001). During pathological oxidative stress, theformation of superoxide radicals often exceeds the endogenous capacityfor inactivation. Furthermore, superoxide stimulates production ofperoxynitrite which nitrates endogenous MnSOD. This protein is nitratedby peroxynitrite in Tyr-34 (Radi, Proc Natl Acad Sci USA 2004;101:4003-4008). Once nitrated, MnSOD looses its enzymatic activity, anevent favoring the accumulation of superoxide and superoxide-drivendamage (Muscoli et al., Br J Pharmacol 2003; 140:445-460).

Recent results indicate that MnSOD inactivation by nitration is an earlyevent in paracetamol-induced hepatic toxicity (Agarwal et al., JPharmacol Exp Ther 2011; 337:110-116). Old results, in addition,indicate that nitration and inactivation of MnSOD are involved inchronic rejection of transplanted kidneys in humans (MacMillan-Crow etal., Proc Natl Acad Sci USA 1996; 93:11853-11858). It may also berelevant to note that actin, which can constitute 5% or more of the cellprotein, is heavily nitrated in sickle cell anemia and that the extentof nitration observed is sufficient to induce cytoskeletalpolymerization (Radi, 2004). Circulating levels of 3-nitrotyrosine mayin addition serve as a biomarker to assess atherosclerosis risks.Furthermore, in addition to atherosclerosis, peroxinitrite and3-nitrotyrosine are believed to be involved in myocardial ischemia,septic and distressed lung, inflammatory bowel disease, amyotrophiclateral sclerosis (Beckman et al., Am J Physiol 1996; 271:C1424-C1437)and diabetes (Fönstermann et al, Br J Pharmacol. 2011; 164:213-223).

Impaired antioxidant defense mechanisms, including reduced SOD activity,and a subsequently increased production of peroxynitrite, may be animportant factor in the pathogenesis of non-alcoholic steatohepatitis(NASH) (Koruk et al., Ann Clin Lab Sci. 2004; 34:57-62). A majorepidemiological and clinical association between either hepatitis B orhepatitis C virus infections and the development of chronic hepatitisand the appearance of hepatocellular carcinoma is evident.Interestingly, peroxynitrite-induced tyrosine-nitration is markedlyincreased in patients with chronic viral hepatitis (Garcia-Monzon etal., J Hepatol. 2000; 32:331-338). Currently, the generally citedmechanism of pathology development in Wilson's disease involvesoxidative damage due to copper overload. Generation of reactive oxygenspecies (ROS) as well as lipid oxidation and DNA damage has beendetected in the liver, particularly at the advanced stages of thisdisease (Burkhead et al., Biometals 2011; 24:455-466).

MnPLED-derivatives are not targets for peroxynitrite and addition ofexogenous MnPLED-derivatives may in such situations re-establish theprotective potential. PLED-derivatives are in addition strong ironbinders, as described in EP 1054670, U.S. Pat. No. 6,310,051 and byRocklage et al., (Inorg Chem 1989; 28:477-485), and someMnPLED-derivatives may have catalase and glutathione reductaseactivities (Laurent et al., 2005), which may further increase theirantioxidant capacity.

For diagnostic imaging use and other sporadic use, dissociation ofmanganese from mangafodipir presents no major toxicological problem. Dueto uptake into CNS, however, for more frequent use, for example intherapeutic methods, accumulated manganese toxicity may represent aserious neurotoxicological problem (Crossgrove et al, NMR Biomed. 2004;17:544-53). Thus, for more frequent therapeutic use, compounds thatreadily dissociate manganese should be avoided and there is a need todevelop means for obtaining desirable therapeutic effects while reducingthe undesirable side effects associated with such therapeutic use.

SUMMARY OF THE INVENTION

The complexes, compositions and methods of the present invention provideimprovements in the preparation and use of metal complexes of PLEDderivatives. In one embodiment, the invention is directed to a mixedmetal complex of a compound of Formula I, or a salt thereof, wherein themixed metals comprise a Group III-XII transition metal and a Group IImetal:

whereinX represents CH or N,each R¹ independently represents hydrogen or —CH₂COR⁵;R⁵ represents hydroxy, optionally hydroxylated alkoxy, amino oralkylamido;each R² independently represents ZYR⁶ wherein Z represents a bond or aC₁₋₃ alkylene or oxoalkylene group, optionally substituted by R⁷;Y represents a bond, an oxygen atom or NR⁶;R⁶ is a hydrogen atom, COOR⁸, alkyl, alkenyl, cycloalkyl, aryl oraralkyl group, optionally substituted by one or more groups selectedfrom COOR⁸, CONR⁸ ₂, NR⁸ ₂, OR⁸, ═NR⁸, ═O, OP(O)(OR⁸)R⁷ and OSO₃M;R⁷ is hydroxy, optionally hydroxylated, optionally alkoxylated alkyl oraminoalkyl group;R⁸ is a hydrogen atom or an optionally hydroxylated, optionallyalkoxylated alkyl group;M is a hydrogen atom or one equivalent of a physiologically tolerablecation;R³ represents a C₁₋₈ alkylene, a 1,2-cykloalkylene, or a 1,2-arylenegroup, optionally substituted with R⁷; andeach R⁴ independently represents hydrogen or C₁₋₃ alkyl, or a saltthereof.

In another embodiment, the invention is directed to a calcium andmanganese complex of a compound of Formula I. The mixed metal complexes,and particularly the mixed calcium-manganese complex calmangafodipirdescribed herein, constitute new chemical entities.

The present invention is also directed to methods of producing a mixedmetal complex which comprises a one-step crystallization from a solutionof the Group III-XII transition metal, the Group II metal, and acompound of Formula I.

In another embodiment, the invention is directed to a method oftreatment of a pathological condition in a patient, comprisingadministering to the patient a mixed metal complex according to theinvention, optionally together with one or more physiologicallyacceptable carriers and/or excipients.

The complexes according to the invention are advantageous in that theGroup II metal stabilizes the complex from releasing the Group III-XIItransition metal. This reduces toxic effects associated with use ofprevious Group III-XII transition metal complexes, for example, MnPLEDderivatives such as mangafodipir. The complexes of the invention mayalso exhibit improved treatment of and/or protection againstpathological conditions, particularly those caused by the presence ofoxygen-derived free radicals, i.e., oxidative stress. Additionalimprovements and advantages of the present invention will be moreapparent in view of the Detailed Description set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description will be more fully understood in viewof the drawings, wherein:

FIG. 1 shows X-ray powder diffraction (XRPD) patterns for threecrystalline forms of a calcium manganese complex of fodipir (DPDP)having an approximate Ca:Mn molar ratio of about 4:1, referred to hereinas “calmangafodipir”, obtained in a one-step crystallization methodaccording to the invention, as described in Example 1.

FIG. 2 shows XRPD patterns of mixtures of calcium fodipir (calfodipir)and manganese fodipir (mangafodipir), rather than a complex according tothe invention; these XRPD patterns indicate all products were amorphousand were shown to absorb water rapidly, as described in Example 2.

FIG. 3 shows the Fourier transform infrared (FT-IR) absorption spectrumof calmangafodipir, lot #7755-C-R0-01-30-01, with the characteristicinfrared absorption bands (wave number) and the correspondingassignments described in Example 3.

FIGS. 4A and 4B show mass spectrum (FIG. 4A) and expanded mass spectrum(FIG. 4B) of calmangafodipir, lot #7755-C-R0-01-30-01 (660-850 m/z), asdescribed in Example 3.

FIG. 5 shows the chemical structure of calmangafodipir, as described inExample 3.

FIG. 6A shows the increase in manganese (Mn) content in 0-24 h urine,expressed as the total content of Mn minus the basal content of Mn, fromrats injected with mangafodipir or calmangafodipir containing 2.59 μmoland 2.52 μmol Mn, respectively. FIG. 6B shows the increase in urinecontent of Mn expressed as percentage of the injected dose. FIG. 6Cshows the increase in zinc content 24 h urine in the same animals.Results are expressed as mean±S.E.M.; n=4 in each group. These figuresare more fully described in Example 4.

FIGS. 7A-7D show the myelosuppressive effects on white blood cells(WBC), lymphocytes (LYM), neutrophils (NEU), and platelets (PLC),respectively, of single intravenous injection of increasing doses (7.5,10.0 and 12.5 mg/kg) of oxaliplatin at 3 and 6 days post injection.Results expressed as mean±S.E.M.; n=5 in each group, as described inExample 5.

FIGS. 8A-8D show WBC, LYM, NEU and PLC, respectively after oxaliplatintreatment alone or in combination with calmangafodipir or mangafodipirin balb/c mice. Controls received vehicle treatment only. Resultsexpressed as mean±S.E.M.; n=5 in each group, as described in Example 5.

FIGS. 9A-9B show the cytotoxic activity of various PLED-derivatives andCaCl₂ at increasing concentrations on colon cancer CT26 cells. Theresults are expressed as mean±S.D.; n=3), as described in Example 6.

FIG. 10A shows the antitumor effect of a high dose oxaliplatin (20mg/kg) in CT26 syngenic balb/c mice in the absence and presence of arelatively high dose of calmangafodipir (50 mg/kg). FIG. 10B shows theantitumor effect of a low dose of oxaliplatin (10 mg/kg in the absenceand presence of a relatively low dose of calmangafodipir. Results areexpressed as mean±S.E.M.; n=10 in vehicle and oxaliplatin 20 mg/kggroups in FIG. 10A; n=5 in all other groups), as described in Example 7.

FIGS. 11A-11C show the Mn content of the brain, pancreas and liver,respectively, after 39 doses of either NaCl (controls), mangafodipir orcalmangafodipir (corresponding in both cases to an accumulated dose of2800 μmol/kg manganese). Results are expressed as mean±S.E.M.; n=17-18in each group, as described in Example 8.

FIGS. 12A-12D show the myelosuppressive effects on white blood cells(WBC), lymphocytes (LYM), neutrophils (NEU), and platelets (PLC),respectively, of single intravenous injection of increasing doses (75,100 and 125 mg/kg) of carboplatin at 3 and 6 days post injection.Results expressed as mean±S.E.M.; n=5 in each group, as described inExample 9.

FIGS. 13A-13D show WBC, LYM, NEU and PLC, respectively after carboplatinin combination with calmangafodipir in balb/c mice. Controls receivedvehicle treatment only. Results expressed as mean±S.E.M.; n=5 in eachgroup, as described in Example 9.

FIG. 14 shows the antitumor effect of a low dose of oxaliplatin (10mg/kg) in CT26 bearing immune competent balb/c mice and in immunedeficient nude balb/c mice (nu/nu) in the absence and presence of arelatively low dose of calmangafodipir. Results are expressed as±S.E.M.; n=5 in each group, as described in Example 10.

FIGS. 15A and 15B show the cytotoxic activity of calmangafodipir andmangafodipir in non-small cell lung cancer cells U1810 and LLC1,respectively. The results are expressed as mean±S.D.; n=3, as describedin Example 11.

The drawings will be more fully understood in view of the Examples.

DETAILED DESCRIPTION

The complexes, compositions and methods of the present invention provideimprovements in the preparation and use of metal complexes of PLEDderivatives, i.e., PyridoxyL EthylDiamine-derivatives, although it isrecognized that the derivatives also act as pro-drugs of PLED as theycan metabolize to form PLED in vivo.

WO 2011/004325 A1 demonstrates how added surplus of fodipir (DPDP) tomangafodipir (MnDPDP) stabilizes it from releasing manganese afteradministration and thereby reduces uptake to CNS, and thereby lowers theneurotoxic potential of mangafodipir considerably. Since it is theintact manganese-containing complex that exerts SOD-mimetic andcytoprotective effects, surplus of fodipir will therefore not only lowerthe neurotoxic potential but it will also increase the cytoprotectiveefficacy considerably.

In vivo-release of manganese from MnPLED-derivatives, includingManganese N,N′-bis-(pyridoxal-5-phosphate)-ethylenediamine-N,N′-diaceticacid (mangafodipir), depends on the presence of free or readilydissociable zinc in the body. Zinc has about 1000 times higher affinitythan manganese for fodipir or its dephosphorylated counterparts(Rocklage et al., 1989). Experimental studies suggest that manganeserelease in vivo from mangafodipir saturates at doses exceeding 5 μmol/kg(Southon et al., 1997). Cardiac and liver imaging with mangafodipir inhealthy volunteers indicates a similar saturation dose in man (Skjold etal., J. Magn. Reson. Imaging 2004; 20:948-952, Toft et al., 1997).

In the invention described in WO 2011/004325 A1, the particulartherapeutic composition was assumed to be obtained by mixing two activepharmaceutical ingredients (APIs), e.g., mangafodipir and fodipir, in aready-to-use solution or administering them separately. It wasdemonstrated that fodipir at a dose level around 5 to 10 μmol/kg had aconsiderable in vivo stabilizing effect on mangafodipir. The firstclinical experiences (Yri et al., 2009 and Karlsson et al., 2011) showthat mangafodipir is therapeutically efficacious at a dose levelsomewhere between 2 and 10 μmol/kg in man. Taking in consideration thehigher efficacy of mangafodipir plus fodipir, it is reasonable topresume that mangafodipir should be therapeutically efficacious inpatients at a dose level close to 1 μmol/kg. This, in turn, teaches usthat a (fodipir+mangafodipir)/mangafodipir ratio close to 5, i.e., aready-to-use formulation containing 4 times more fodipir thanmangafodipir, should be efficacious. This furthermore suggests aready-to-use formulation containing 40 mM fodipir and 10 mMmangafodipir-administration of 0.1 to 0.2 ml of this formulation per kgbody weight—would result in a dose of 1 to 2 μmol/kg mangafodipir and 4to 8 μmol/kg fodipir.

Calcium has about 10⁹ times lower affinity for fodipir than zinc andabout 10⁶ times lower affinity for fodipir than manganese. However,taking in consideration that calcium is present in much higherextracellular concentrations than zinc and manganese, rapid intravenousbolus administration of fodipir may induce acute reduction in theextracellular concentration of free calcium. Since the heart isabsolutely dependent on extracellular calcium for its blood pumpingactivity, reduction in the extracellular content of free calcium may inturn induce acute heart failure. However, as discussed in WO 2011/004325A1 this problem can be easily solved by making use of thecalcium-complexed DPDP, i.e., CaDPDP.

Surprisingly, it has been discovered that CaDPDP may be employed in acomplex with manganese in PLED-derivatives. Further, surprisingly,complexes of calcium and manganese, for example, calmangafodipir, andcomplexes of other Group II metals and Group III-XII transition metals,can be obtained.

Thus, in accordance with one aspect, the invention is directed to amixed metal complex of a compound of Formula I, or a salt thereof,wherein the mixed metals comprise a Group III-XII transition metal and aGroup II metal:

whereinX represents CH or N,each R¹ independently represents hydrogen or —CH₂COR⁵;R⁵ represents hydroxy, optionally hydroxylated alkoxy, amino oralkylamido;each R² independently represents ZYR⁶ wherein Z represents a bond or aC₁₋₃ alkylene or oxoalkylene group, optionally substituted by R⁷;Y represents a bond, an oxygen atom or NR⁶;R⁶ is a hydrogen atom, COOR⁸, alkyl, alkenyl, cycloalkyl, aryl oraralkyl group, optionally substituted by one or more groups selectedfrom COOR⁸, CONR⁸ ₂, NR⁸ ₂, OR⁸, ═NR⁸, ═O, OP(O)(OR⁸)R⁷ and OSO₃M;R⁷ is hydroxy, optionally hydroxylated, optionally alkoxylated alkyl oraminoalkyl group;R⁸ is a hydrogen atom or an optionally hydroxylated, optionallyalkoxylated alkyl group;M is a hydrogen atom or one equivalent of a physiologically tolerablecation;R³ represents a C₁₋₈ alkylene, a 1,2-cycloalkylene, or a 1,2-arylenegroup, optionally substituted with R⁷; andeach R⁴ independently represents hydrogen or C₁₋₃ alkyl.

As used herein the terms “alkyl” and “alkylene” include straight-chainedand branched, saturated and unsaturated hydrocarbons. The term“1,2-cykloalkylene” includes both cis and trans cycloalkylene groups andalkyl substituted cycloalkylene groups having from 5-8 carbon atoms. Theterm “1,2-arylene” includes phenyl and naphthyl groups and alkylsubstituted derivatives thereof having from 6 to 10 carbon atoms. Unlessotherwise specified, any alkyl, alkylene or alkenyl moiety mayconveniently contain from 1 to 20, more specifically 1-8, morespecifically 1-6, and even more specifically, 1-4 carbon atoms.Cycloalkyl moieties may conveniently contain 3-18 ring atoms,specifically 5-12 ring atoms, and even more specifically 5-8 ring atoms.Aryl moieties comprising phenyl or naphthyl groups are preferred. Asaralkyl groups, phenyl C1-8 alkyl, especially benzyl, are preferred.Where groups may optionally be substituted by hydroxyl groups, this maybe monosubstitution or polysubstitution and, in the case ofpolysubstitution, alkoxy and/or hydroxyl substituents may be carried byalkoxy substituents.

The compound of Formula I may have the same or different R² groups onthe two pyridyl rings and these may be attached at the same or differentring positions. In a specific embodiment, the substitution is at the 5-and 6-positions, or more specifically, the 6-position, i.e. para to thehydroxyl group. In a specific embodiment, the R² groups are identicaland identically located, and more specifically are in the6,6′-positions. In yet more specific embodiments, each R⁶ is a mono- orpoly(hydroxy or alkoxylated) alkyl group or a group of the formula OP(O)(OR⁸)R⁷.

In another embodiment, the invention is directed to a calcium andmanganese complex of a compound of Formula I. In one embodiment, R⁵ ishydroxy, C₁₋₈ alkoxy, ethylene glycol, glycerol, amino or C₁₋₈alkylamido; Z is a bond or a group selected from CH₂, (CH₂)₂, CO, CH₂CO,CH₂CH₂CO and CH₂COCH₂; Y is a bond; R⁶ is a mono- or poly(hydroxy oralkoxylated) alkyl group or of the formula OP(O)(OR⁸)R⁷; and R⁷ ishydroxy, or an unsubstituted alkyl or aminoalkyl group. In a morespecific embodiment, R³ is ethylene and each group R¹ represents—CH₂COR⁵ in which R⁵ is hydroxy. In a further embodiment, the compoundof Formula I isN,N′-bis-(pyridoxal-5-phosphate)-ethylenediamine-N,N′-diacetic acid(DPDP), or a pharmaceutically acceptable salt thereof. In still furtherembodiments, the pharmaceutical matter is a mixed manganese and calciumcomplex ofN,N′-bis-(pyridoxal-5-phosphate)-ethylenediamine-N,N′-diacetic acid, ora salt thereof.

The mixed metal complex may include any combination of metals from theindicated Groups. In a specific embodiment, the Group III-XII transitionmetal is Mn²⁺, Cu²⁺, Fe²⁺ and/or Ni²⁺ and the Group II metal is Ca²⁺and/or Mg²⁺. In a more specific embodiment, the Group III-XII transitionmetal is Mn²⁺ and the Group II metal is Ca²⁺ or a mixture of Ca²⁺ andMg. In more specific embodiments, the Group II metal is a mixture ofCa²⁺ and Mg²⁺ in a Ca²⁺/Mg²⁺ molar ratio of about 0.1-50, morespecifically about 0.1-10.

In further embodiments, the mixed metal complex contains a molar ratioof Group II metal to Group III-XII transition metal of about 1-10. In aspecific embodiment, the Group III-XII transition metal is Mn²⁺ and theGroup II metal is Ca²⁺ and the molar ratio of Ca²⁺/Mn²⁺ is about 4. In amore specific embodiment, the Group III-XII transition metal is Mn²⁺ andthe Group II metal is Ca²⁺ and the molar ratio of Ca²⁺/Mn²⁺ is about 4,and the compound of Formula I is DPDP, i.e., the complex is the compoundcalmangafodipir as described herein.

In another aspect, the invention is directed to a method of producing amixed metal complex of the invention, in one preparation/crystallizationstep. The method comprises a one-step crystallization from a solution ofthe Group III-XII transition metal, the Group II metal, and a compoundof Formula I. In a specific embodiment, one-step crystallization from asolution of manganese, calcium, and a compound of Formula I isconducted. In a more specific embodiment, the solution has with a(Ca+Mn)/Mn ratio close to (4+1)/1=5, i.e., with a compositionstoichiometry close to Ca₄Mn(DPDP)₅.

X-Ray Powder Diffraction (XRPD) is most widely used in theidentification and characterization of crystalline solids, each of whichproduces a distinctive diffraction pattern. Both the positions(corresponding to lattice spacings) and the relative intensity of thelines are indicative of a particular phase and material, providing a“fingerprint” for comparison. As shown in the Examples, an XRPD analysisof calmangafodipir demonstrates without doubt that calmangafodipir isone chemical entity, i.e., a complex, rather than a simple blend, seeExample 1. FIG. 1 shows a stacked plot of the three crystalline forms ofcalmangafodipir which interconvert according to the ambient humidity.Variable humidity XRPD analysis demonstrated Form B to be stable over40% relative humidity (RH), Form A is stable at 0-10% RH, and Form C isstable between 6-36% RH. Mixtures of Forms B and C were observed between38-44% RH, and form conversions were observed to occur within 3 hours ona 10 mg scale.

The one step preparation can be performed with or without seeding, butseeding (as exemplified in Example 1) allows for better control of thecrystallization.

The above briefly mentioned one step preparation is superior to that ofmixing individual metal complexes. Thus, in a specific embodiment, thecomplex of calcium and manganese is a crystalline material and readilydistinguishable from a simple mixture of mangafodipir (MnDPDP) andcalfodipir (CaDPDP) in the desirable amounts, as shown in Example 2.

The Examples also show improvements and advantages of the complexesaccording to the invention, as represented by the calcium-manganesecomplex calmangafodipir. Regarding the in vivo manganese stability,calmangafodipir is at least as stable as a true mixture of mangafodipirand fodipir, as demonstrated in Example 4. This will result insignificantly less retention of manganese in the brain, as demonstratedin Example 8. Since the cytoprotective efficacy mainly depends on theintact manganese complex mangafodipir or its dephosphorylatedcounterparts, MnDPMP and MnPLED, the efficacy of calmangafodipir issuperior to that of mangafodipir, as exemplified in Example 5.Furthermore, mangafodipir has surprisingly been found to possesscytotoxic effects against cancer cells, e.g., CT26 cells (Laurent etal., 2005; Alexandre et al., 2006; EP 16944338). However, as describedin WO 2009/078794 A1 and in Kurz et al., 2012, this is an inherentproperty of fodipir alone or of its dephosphorylated counterparts, DPMPand PLED, and not of the intact metal complex mangafodipir or itsdephosphorylated counterparts, MnDPMP and MnPLED. In Example 6 it isshown that fodipir is about 20 times more efficacious than mangafodipirin killing CT26 colon cancer cells, and Example 11 shows thatcalmangafodipir is about 28 times more efficacious than mangafodipir inkilling U1810 non-small cell lung cancer cells. Dissociation to someextent of manganese from fodipir, under in vitro conditions, mostprobably explains the cancer killing efficacy of mangafodipir.Calmangafodipir as described in Examples 1, 2 and 3, as compared withmangafodipir at manganese equimolar concentrations, is on the other handas efficacious as fodipir alone, i.e., the killing efficacy ofcalmangafodipir is much higher than that of mangafodipir at equimolarmanganese concentrations. This finding suggests two importantproperties. Firstly, dephosphorylated PLED is probably as efficacious asits phosphorylated counterpart fodipir with respect to its cancer cellkilling ability, and secondly, the lower stability of MnPLED incomparison to that of mangafodipir (Rocklage et al., 1989) probablyexplains the higher efficacy of MnPLED. The lack of any cytotoxicactivity of ZnDPDP and ZnPLED is due to the 1000 times higher stabilityof these complexes in comparison to their manganese counterparts(Rocklage et al., 1989).

During the development of mangafodipir as an MRI contrast agent, it wasdiscovered that MnDPDP caused fetal skeletal abnormalities in rats butnot in rabbits. Importantly, this teratogenic effect seen in rats is notcaused by intact mangafodipir per se but by dissociated manganese (Grantet al., Acta Radiol. 1997; 38:759-769). Although it is uncertain whethermanganese will cause skeletal abnormalities in the human fetus, it is ofcourse essential to protect potentially pregnant women from beingexposed to mangafodipir. This represents a minor problem for mostclinical applications but a major occupational problem, in particularduring the production where costly measures have to be taken in order toprotect fertile and potentially pregnant women from exposure tomangafodipir. Importantly, the fact that calmangafodipir will releasemanganese to a much lesser extent than mangafodipir after beingaccidentally absorbed into the body will of course reduce the riskconsiderably that a fetus develops skeletal malformations. Secondly,since calmangafodipir is significantly more efficacious thanmangafodipir at equimolar doses of manganese, the need of manganese isconsiderably reduced for every dose of calmangafodipir produced, whichwill result in less manganese exposure during production.

Furthermore, the preparation of a single active pharmaceuticalingredient reduces the cost of manufacture of a treatment dosage. Inaddition, the need for dosing of a single material reduces the chancefor errors in the formulation of the product. In stability testing, thestability of a crystalline product has been shown to be superior toamorphous material, such as that formed by spray drying a mixture of thetwo API's. As illustrated in Example 2, amorphous material obtained fromspray drying was shown to absorb water rapidly, forming fused particlesand/or sticky solids within 24 h exposure at 25° C./60% RH and 40°C./75% RH. In contrast, crystalline calmangafodipir remained a freeflowing solid even after 7 days under the same exposure conditions.

It has been discovered, as described in WO 2011/004325 A1, that additionof surplus non-manganese-containing PLED derivative, for example DPDP,to MnPLED-derivative therapy, protects mangafodipir from releasingneurotoxic manganese in vivo. Although the mechanism behind manganeseuptake into the brain is not entirely understood, surplus of thenon-manganese-containing PLED derivative such as fodipir administered incombination with the manganese-containing PLED derivative such asmangafodipir significantly reduces the uptake of manganese to the brain.While not wishing to be bound by theory, it is believed that thecombination according to the invention of WO 2011/004325 A1 maintainsthe MnPLED chelator form, whereby increased amounts of chelates areavailable for excretion and the amount of free Mn for uptake into thebrain and other organs is reduced. Low molecular weight manganesechelates, like MnPLED-derivatives, and their Zn-counterparts willreadily be excreted through the kidney, governed by the glomerulusfiltration rate (GFR), whereas manganese not bound to a low molecularweight chelator will be retained for quite a while in the body andexcreted slowly and mainly via the biliary route (Toft et al., 1997). Asshown in the following Example 8 herein, repeated (39 times over 3months) intravenous injections of a high dose (36 times the assumedclinical assumed dose) of calmangafodipir into rats caused significantlyless retention of manganese in the brain, compared to that caused bymangafodipir. The total dose in both cases corresponded to approximately2800 μmol/kg of manganese. This example also shows that the pancreastakes up and retains dissociated manganese to a relatively large extent,a property previously described by Ni et al. (Acta Radiol 1997;38:700-707) and utilized as a promising diagnostic MRI method of thepancreas (Ahlström et al, Acta Radiol 1997; 38:660-664). Thesignificantly lower manganese level in the pancreas ofcalmangafodipir-treated rats in comparison to those treated withmangafodipir further confirms the improved toxicological profile ofcalmangafodipir. Although, the Mn content of the liver was statisticallysignificant elevated in the mangafodipir group, the relative elevationwas much less than those in the brain and pancreas. A single diagnosticdose of mangafodipir (5 μmol/kg b.w.) is known to cause rapid increasein the Mn content of both the pancreas and the liver of rats—after 2hours the Mn content of the pancreas was approximately 10 times higherthan the basal value, and the corresponding value of the liver wasincreased about 2 times (Ni et al., 1997). Whereas Ni et al found the Mncontent still elevated after 24 hours in the pancreas (about 5 times thebasal value), it was back to baseline in the liver at that time point.This presumably reflects the high capacity of the liver to handlemanganese and its important physiological role in manganese homeostasis.This is further supported by the present results showing just a modestincrease in liver Mn after heavy exposure to mangafodipir. The improvedtoxicological profile of calmangafodipir is clearly illustrated byExample 8 herein.

When a clinical dose of a MnPLED-derivative such as mangafodipir (i.e.,5-10 μmol/kg b.w., intravenous administration) is used as an MRIcontrast agent in a human, about 80% of the manganese bound to fodipir(DPDP) is exchanged with zinc (Toft et al., 1997). As smaller doses ofmangafodipir are administered, the percentage of manganese whichdissociates will be even greater. Mangafodipir behaves in thatperspective in a similar manner in rats and dogs (Hustvedt et al.,1997); however, almost all manganese in mangafodipir is exchanged forzinc when the compound is administered into pigs and is hence withoutcytoprotective effects in pigs (Karlsson et al., 2001). On the otherhand, administration of low doses of MnPLED causes profoundcytoprotective effects in pigs, seen as a significantly reducedmyocardial infarct size upon ischemia-reperfusion. Although the reportedstability constant between Mn²⁺ and PLED is considerably lower than thecorresponding figure for Mn²⁺ and fodipir (Rocklage et al., 1989),MnPLED for some unknown reason escapes metal exchange. Displacement ofmanganese is a prerequisite and therefore desirable for use as an MRIcontrast agent, e.g., for liver and pancreas diagnostic purposes.However, the SOD-mimetic therapeutic effect against various forms ofoxidative stress depends fully on the intact manganese PLED-derivativecomplex (Brurok et al., 1999; Karlsson et al., 2001). For example,whereas in vivo administration of mangafodipir protects against variousoxidative stressors, e.g., ischemia-reperfusion, cytotoxic/cytostaticdrugs and acetaminophen intoxication, it does not protect the pig heartagainst ischemia-reperfusion-induced myocardial infarction (Karlsson etal., 2001), results from which it can be concluded that the in vivocytoprotective effects of MnPLED-derivatives are an inherent property ofthe intact manganese complex.

The presence of Ca in an approximately 4 times excess to Mn, as incalmangafodipir, profoundly stabilizes the complex or itsdephosphorylated counterparts from releasing manganese after injectionsand thus provides another important advantage, namely increasedtherapeutic efficacy. For example, when a clinically relevant imagingdose of MnDPDP (5-10 μmol/kg) is intravenously injected, about 80% ofthe manganese originally bound to DPDP is released, contributing to theimaging efficacy. Consequently less than 20% remains bound to DPDP orits dephosphorylated counterparts, contributing to the therapeuticactivity of MnDPDP. As the release of manganese from the complex can beeffectively reduced by an approximately 4 times excess of Ca, incomparison to Mn, in calmangafodipir, this means that manganese can bereduced considerably for an equipotent therapeutic effect, as clearlyexemplified in the present invention. At lower and, in certainembodiments, more therapeutic relevant doses, the stabilizing effect ofCa will even be more accentuated. This, in turn, means that the use ofcalmangafodipir in comparison to mangafodipir will have profound effectson the neurotoxic potential—both as a result of an in vivo stabilizingeffect and as a result of increased therapeutic efficacy of the newpharmaceutical matter calmangafodipir, with a composition stoichiometryclose to Ca₄Mn(DPDP)₅.

Zinc is present in all body tissues and fluids. The total body zinccontent in humans has been estimated to be 2-3 g (Folin et al.,BioMetals 1994; 7:75-79). Plasma zinc represents about 0.1% of totalbody zinc content, and it is mainly this small fraction of zinc thatcompetes with manganese for binding to fodipir or its dephosphorylatedcounterparts, DPMP and PLED, after administration. The human body has avery high capacity to maintain zinc homeostasis through synergisticadjustments in gastrointestinal absorption and excretion (King et al., JNutr 2000; 130:1360S-1366S).

While not wishing to be bound by theory, from preclinical work (Southonet al., 1997) and from clinical work (Skjold et al., 2004), it may bereasonable to assume that the body contains 5 to 10 μmol/kg body weight(b.w.) zinc that is readily exchangeable for manganese in aMnPLED-derivative such as mangafodipir. This substantially correspondsto the zinc content of the plasma as described above. ThePLED-derivatives such as fodipir contain one binding site formanganese/zinc per molecule. Thus, in view of the 1000 times higheraffinity for zinc to the chelator, the presence of calcium incalmangafodipir at a ratio of around 4 in comparison to manganese willprotect against release of manganese after parenteral administration toa patient.

In another embodiment, the invention is directed to methods fortreatment of a pathological condition in a patient, including, but notlimited to, a pathological condition caused by the presence ofoxygen-derived free radicals, i.e., oxidative stress, by administrationof the complex. In a specific embodiment, the pathological condition iscaused by superoxide resulting in subsequent lipid peroxidation and/orprotein nitration. In a specific embodiment, the complex may beadministered for therapeutic treatment of such a pathological conditionin a human patient or another mammal. In another specific embodiment, acomplex according to the invention is administered for treatment of apathological condition caused by the presence of oxygen-derived freeradicals, i.e., oxidative stress, in a mammal.

In one embodiment, the complex is employed in cytotoxic or cytostaticdrug treatment, wherein the complex is administered to provideprotection from disadvantageous side effects of thecytotoxics/cytostatic drugs, for example, one or more cancer drugs incancer patients. In a more specific embodiment, the cytotoxic orcytostatic drug comprises at least one of doxorubicin, epirubicin,oxaliplatin, carboplatin, cisplatin, 5-fluorouracil, docetaxel orpaclitaxel. In additional embodiments, the pathological condition ismyeolosuppression or neurotoxicity, or both.

The methods according to the invention may also include, but are notlimited to, treatment of acetaminophen-induced liver failure,non-alcoholic steatohepatitis (NASH), viral-induced chronic hepatitis,Wilson's disease, diabetes, ischemic heart disease, includingischemia-reperfusion-induced injury, or myocardialischemia-reperfusion-induced injury, both in an acute as well aselective setting, a condition associated with a thrombolytic treatment,a cardiopulmonary bypass, or percutaneous transluminal angioplasty, oris a result of cardiac or organ transplantation surgery or stroke. Inadditional embodiments, the methods according to the invention may alsoinclude treatment of iron-related conditions, including iron overload,for example, thalassemia, sickle cell anemia or transfusionalhemosiderosis, hepatitis-induced liver cirrhosis, radiation inducedinjury, for example, resulting from radiation therapy, variousneurodegenerative diseases, including Alzheimer's disease, amyotrophiclateral sclerosis (ALS), Parkinson's disease, and multiple sclerosis,and the like.

In yet further embodiments, the methods according to the invention areadministered as replacement therapy for a pathological condition of lowmanganese superoxide dismutase (MnSOD) activity, such as occurs invarious forms of cancer (Buettner, Anticancer Agents Med Chem. 2011;11:341-346). Thus, in one embodiment, the invention is directed to amethod for the treatment of non-small cell lung cancer by administeringthe complex of the invention, or, more specifically, calmangafodipir. Inadditional embodiments, the invention is directed to a method for thetreatment of colorectal cancer, prostate cancer, breast cancer,pancreatic cancer, or malignant melanoma, by administering the complexof the invention, or, more specifically, calmangafodipir.

An interesting and probably important property for the antitumor effectof mangafodipir may be its lymphocyte protecting property, as shown inExample 5, and by Laurent et al., 2005 and Alexandre et al., 2006.Although inflammatory processes secondary to oxidative stress damagenormal tissue, they may in fact be beneficial to tumor tissue bycreating growth factor-rich microenvironment and promoting growth ofcancerous clones (Anscher, Onclogist 2010; 15:350-359; Kareva, TranslOncol 2011; 4:266-270; Kerkar et al., Cancer Res 2012; 72:3125-3130). Astriking example is the existence of tumor-associated macrophages thataccumulate preferentially in the poorly vascularized regions of tumorsand secrete cytokines that actually promote tumor growth. Moreover, notonly can these cytokines promote tumor growth but they have also beenshown to suppress activation of CD8+ T-lymphocytes that are mostefficient in tumor elimination. In fact, there is an increasing interestfor the importance of T-lymphocyte-mediated immune response for theoutcome of cancer chemotherapy (Zitvogel et al., Nat Rev Clin Oncol2011; 8:151-160; Kerkar et al., 2012). It is known that severelymphopenia (<1000 cells/μl) negatively affects the chemotherapyresponse. A collection of mouse cancers, including CT26 colon cancer,MCA205 fibrosarcomas, TSA cell-line breast cancers, GOS cell-lineosteosarcomas and EL4 thymonas, respond to chemotherapy with doxorubicinand oxaliplatin much more efficiently when they are implanted insyngenic immune-competent mice than in immune-deficient hosts, i.e.,nude mice (Zitvogel et al., 2011). This is in line with clinical studiesrevealing that IFN-γ-producing CD8+ T-lymphocytes are potent cancerimmune effectors. Furthermore, a high neutrophil/lymphocyte ratio isassociated with a low overall survival for patients with advancedcolorectal cancer (Chua et al., Br J Cancer 2011; 104:1288-1295). Takinginto consideration that mangafodipir and in particular calmangafodipirare highly efficient lymphocyte-protecting agents during chemotherapy,it is plausible that this property is of particular importance during invivo conditions.

To be clinically useful, a chemotherapy protectant or a radiotherapyprotectant used in cancer patients should fulfill three criteria: (i)the agent should protect normal tissue fromchemotherapy/radiotherapy-induced toxicity but not protect tumor tissue(at least not to any greater extent)—otherwise no benefit will beobtained; (ii) the agent should be delivered with relative ease and withminimal toxicity; and (iii) the agent should protect normal tissuesagainst dose-limiting toxicities or those responsible for significantreduction in quality of life (Citrin et al., 2010). The compounds of theinvention, and calmangafodipir in particular, fulfill all thesecriteria, as the examples herein demonstrate. The reason whymangafodipir and calmangafodipir protect nonmalignant cells but damagecancer cells is seemingly a paradox. While not wishing to be bound bytheory, it may, however, be that protection of nonmalignant cells andcytotoxic actions against cancer cells just are two sides of the samecoin. An elevated oxidative status is indispensable for mitogenicstimulation in transformed cells (Irani et al., Science 1997;275:1649-1652). A number of studies have reported that reactive oxygenspecies (ROS) play an important role in promoting tumor metastasis(e.g., Behrend et al., Mol Cell Biol 2005; 25:7758-7769). These data areconsistent with a large body of literature suggesting that the redoxbalance of many epithelial tumor cells favors an elevated oxidant setpoint (Doroshow, 2006), including CT26 cells (Laurent et al., 2005;Alexandre et al., 2006). MnSOD suppresses cell growth in a variety ofcancer cell lines and in mouse models. Furthermore, overexpression ofMnSOD induced growth arrest in the human colorectal cancer cell lineHCT116 and increased senescence that required the induction of p53(Behrend et al., 2005). Introduction of the normal MnSOD gene in cancercells alters the phenotype and the cells lose their ability to formcolonies in culture and tumors in nude mice (Church et al., Proc NatlAcad Sci USA 1993; 90:3113-3117). The elevated oxidative status seen incancer cells typically leads to increased production of .O₂ ⁻ whichreadily reacts with .NO to form highly toxic ONOO⁻ resulting in tyrosinenitration, the “ugly” side of .NO (Beckman et al, 1996; Radi, 2004).Interestingly, convincing evidence suggests that tyrosine nitration isinvolved in the above described suppression of CD8+ lymphocyte mediatedimmunological response in tumors of (Bronte et al., J Exp Med 2005; 201,1257-1268; Molon et al., J Exp Med 2011; 208:1949-1962). It may be thatcalmangafodipir through its SOD mimetic activity inhibits ONOO⁻production and hence the immunological suppression, explaining theincreased antitumor effect seen in immune competent mice but not immuneincompetent mice, as demonstrated by Example 10. Peroxynitrite is notcapable of protein nitration directly but typically needs a redox activetransition metal like iron or copper (and even manganese may fulfillthis need) (Radi, 2004). Fodipir and its dephosphorylated metaboliteshave an extremely high affinity for Fe³⁺ (Rocklage et al., 1989). Thisproperty may, in addition to the SOD mimetic activity ofcalmangafodipir, be of a particular importance for the antitumoractivity. In addition to the T-lymphocyte-dependent action, the directimmune-independent action of DPDP and PLED, can be due to inhibition oftopoisomerase II, as a recent paper of Kurz (2012) may suggest.

The complex may be administered in a pharmaceutical composition.Optionally, the pharmaceutical compositions of the present invention mayinclude one or more physiologically acceptable carriers and/orexcipients, in a manner well-known to those skilled in the art. In oneembodiment, the complex may for example be suspended or dissolved in aliquid medium, optionally with the addition of pharmaceuticallyacceptable excipients. Suitable excipients for the pharmaceuticalcompositions include any conventional pharmaceutical or veterinaryformulation excipients, including, but not limited to, stabilizers,antioxidants, osmolality adjusting agents, buffers, pH adjusting agents,binders, fillers, and the like. The pharmaceutical compositions may bein a form suitable for administration, including both parenteral andenteral administration. In a specific embodiment, the composition is ina form suitable for example injection or infusion. Thus, thepharmaceutical compositions of the present invention may be in aconventional pharmaceutical administration form such as a tablet,capsule, powder, solution, suspension, dispersion, syrup, suppository,aerosol, ointment, plaster, or the like. In a further embodiment, thecomplex is in freeze dried form and, if desired, may be reconstitutedprior to administration. The freeze dried complex may be in a freezedried composition containing one or more stabilizers, and/or otherexcipients known for use in freeze drying.

Such compositions according to the present invention may be administeredby various routes, for example orally, transdermally, rectally,intrathecally, topically or by means of inhalation or injection, inparticular subcutaneous, intramuscular, intraperitoneal or intravascularinjection. Other routes of administration may be used as well, includingintratympanic and intranasal, and routes which increase theeffectiveness, the bioavailability or the tolerance of the products arepreferred. The most appropriate route can be chosen by those skilled inthe art according to the particular formulation which is used. Suitabledosages will be apparent relative to the selected treatment. In oneembodiment, the treatment method according to the invention comprisesadministering about 0.01 to 50 μmol/kg body weight of the mixed metalcomplex. In more specific embodiments, the treatment method according tothe invention comprises administering about 0.1 to 10 μmol/kg, or about0.1 to 5 μmol/kg, body weight of the mixed metal complex.

The following examples demonstrate various embodiments and aspects ofthe invention.

Example 1 Method

A jacketed 100-L reactor—flushed with N₂— was charged with fodipir(DPDP) (4.0 kg anhydrous based, 6.27 mol, 1 equiv) and deionized (DI)water (19.2 L, 4.88 vol). The pH of the batch was adjusted to 5.7 withdilute NaOH (8.9 L total, 17.5 mol NaOH, 2.8 equiv; prepared from 1.41kg of 50 wt % NaOH and 8.0 L of DI water) over 35 min (21.0-23.3° C.;external cooling). The slurry was stirred for 1 h at 20-25° C. duringwhich time a solution formed. To this was sequentially charged Ca(OH)₂(361.1 g, 4.87 mol, 0.78 equiv), L-ascorbic acid (55.1 g, 0.313 mol, 5mole %), and MnO (80 g, 1.13 mol, 0.18 equiv). After the addition ofeach reagent the batch was stirred for 30-60 min at 20-25° C. and the pHwas measured (after Ca=6.24, slight cloudy pale yellow to rust; afterascorbic=6.28 less cloudy rust; after Mn=6.38, cloudy rust toyellow-green). The cloudy batch was stirred for 16 h at 20-25° C., pHwas measured (6.36) and the batch was filtered through a 0.3μ in-linefilter into a clean 100-L reactor. Meanwhile, an ethanol (EtOH) 23Asolution was prepared with acetone (5.9 L, 1.47 vol) and EtOH (74 L,18.5 vol). A portion of the EtOH 23A solution (8.0 L, 2 vol) was chargedto the batch over 30 minutes at 20-25° C. during which time the solutionbecame cloudy. The batch was seeded with calmangafodipir (40 g, 1 wt %)and stirred for 30 min at 20-25° C. to ensure solids persisted.

The batch temperature was adjusted to 15° C. over the course of 1 h andthen aged for 30 min at 13.8-15.5° C. To the batch was charged EtOH 23A(56 L, 14 vol) over 10 h (11-14° C.). The slurry was mixed for 13 h at5-10° C. and then filtered to collect the solids. The reactor and solidswere rinsed with chilled (0-10° C.) EtOH 23A (14 L, 3.5 vol), the solidswere conditioned for 2 h and then dried in a vacuum oven at 45° C. for72 h to afford 4.819 kg (93% yield adjusted for water content) ofcalmangafodipir (lot #11AK0105B) as a yellow solid. HPLC analysis showeda purity of 98.8%. Oven Karl Fisher analysis (@170° C.) showed 10.1%water. ICP analysis indicated 4.27% Ca, 1.37% Mn, 8.64% Na for a Ca/Mnratio of 4.27, i.e. with a composition stoichiometry close toCa₄Mn(DPDP)₅.

Results

XRPD (X-Ray Powder Diffraction) analysis was conducted and the resultingpatterns are shown in FIG. 1. The XRPD patterns demonstrate thatcalmangafodipir is a single chemical entity rather than a simple blend.FIG. 1 shows a stacked plot of the three known crystalline forms ofcalmangafodipir which interconvert according to the ambient humidity.Variable humidity XRPD analysis demonstrated Form B to be stable over40% RH, Form A stable at 0-10% RH, and Form C stable between 6-36% RH.Mixtures of Forms B and C were observed between 38-44% RH, and formconversions were observed to occur within 3 hours on a 10 mg scale.

Conclusion

The XRPD demonstrated surprisingly calmangafodipir as being one chemicalentity rather than a simple blend (FIG. 1).

Example 2 Method

In a 4:1 ratio approximately 200 mg of calfodipir (CaDPDP) and 50 mg ofmangafodipir (MnDPDP) were weighed into a 40 mL vial and dissolved in 40mL of DI water at room temperature to generate a yellow solution. Theyellow aqueous solution was spray dried using a Buchi Mini-Spray DryerB-290 while attached to the Buchi dehumidifier B-296 as an airconditioner allowing the intake of air from the laboratory. Spray dryingoptimization experiments were performed at elevated inlet temperatures(220° C.) with varying feed rates (20, 30, 40, 50 and 60%). Recovery ofproduct ranged from 180-230 mg.

Results

The resulting materials were analyzed by XRPD (FIG. 2) which indicatedall products were amorphous and were shown to absorb water rapidly,forming fused particles and/or sticky solids within 24 h exposure at 25°C./60% RH and 40° C./75% RH.

Example 3

This example elucidates the structure of the new chemical entitycalmangafodipir by making use of: Infrared Absorption Spectroscopy; MassSpectroscopy; and Elemental Analysis. NMR analysis cannot be utilizedfor analysis of calmangafodipir due to the paramagnetic nature of Mn.

Methods

The analyses described in this example were conducted on a productproduced according to the one step method described herein. The productis identified as lot#7755-C-R0-01-30-01, and was prepared essentially asdescribed in Example 1. A portion of this product has been certified asa reference standard for calmangafodipir.

Infrared Absorption Spectroscopy.

The Fourier transform infrared (FT-IR) absorption spectrum ofcalmangafodipir, lot #7755-C-R0-01-30-01 was obtained using attenuatedtotal reflection (ATR) on a Thermo-Nicolet Avatar 370 spectrometer.

Mass Spectroscopy.

The mass spectrum of calmangafodipir, lot #7755-C-R0-01-30-01, wasacquired on a Waters Q-Tof Micro MS/MS system. Electrospray Ionization(ESI) (positive ion polarity mode) was chosen for the MS analysis. Thesample was dissolved in a solution of 50:50 Acetonitrile/Water+0.1%Formic Acid at a concentration of 10 μg/mL. The solution was infuseddirectly into the source at a rate of 10 μL/minute.

Elemental Analysis.

Calmangafodipir lot #7755-C-R0-01-30-01 was manufactured using a Ca/Mnmolar ratio of 4.26 and 2.8 mol Na/mole of fodipir. The theoreticalmetals content of the complex with this composition is 1.41% Mn, 4.38%Ca, and 8.69% Na.

Results

The infrared absorption spectrum is shown in FIG. 3, and thecharacteristic infrared absorption bands (wave number) and thecorresponding assignments are as follows:

Wave number (cm⁻¹) Assignment 3250 N—H 3027-2848 C—H 1581 C═O 1528 C═C1471, 1437 CH₂, CH₃ 1383 CH₃ 1277 P═O 1092 P—OH 1038, 977, 933, 913P—O—C 827, 813, 770, 751 Aromatic C—H

The mass spectrum and an expanded mass spectrum of the sample are shownin FIGS. 4A and 4B, respectively. The spectrum presents as that ofcalfodipir and mangafodipir superimposed on each other. The exact massof fully protonated calfodipir is 676 and a mass of 677 for [M+1] isobserved. The exact masses for monosodium, disodium, trisodium, andtetrasodium are 698, 720, 742, and 764, respectively. The spectrum shows[M+1] for each species at 699, 721, 743, and 765, respectively. Theexact mass of fully protonated Mangafodipir is 691, with thecorresponding monosodium, disodium trisodium, trisodium, and tetrasodiumspecies at 713, 735, 757, and 779, respectively. The spectrum exhibitsmasses at 692, 714, 736, 758, and 780 for [M+1] for each species.

The metals content results for lot#7755-C-R0-01-30-01 were 1.48% Mn,4.44% Ca, and 8.56% Na and are in agreement with the expected values,confirming that both the manganese and calcium are complexed, withsodium as the counterion, and that little or none of the calcium ispresent simply as a counterion.

Conclusion

These results are consistent with the structure shown in FIG. 5. FIG. 5shows the ideal and generic 4:1 Ca/Mn with 3 Na as counterions, whichgives the molecular weight of 745.43 shown in FIG. 5. The averagemolecular weight for material prepared as described and studied in thisExample has a 4.26 Ca/Mn ratio and 2.8 Na as counterions, having amolecular weight of 740.89.

Example 4

This example measured manganese (Mn) and zinc (Zn) urine excretion inanimals receiving calmangafodipir or mangafodipir, at Mn equimolardoses.

Method

Eight male Wistar rats (approximately 250 g) were injectedintravenously, via one of the tail veins, with 0.25 ml of a 50 mMcalmangafodipir (lot #11AK0105B) solution, containing approximately 10mM Mn and 40 mM Ca, or 0.25 ml 10 mM mangafodipir (lot #02090106),containing 10 mM Mn. After injection, the rats were immediately placedin metabolic cages for urine collection over a period of 0-24 hours. Toobtain basal content of manganese (Mn) and zinc (Zn) in urine, twoadditional (control) rats received 0.25 ml saline and were placed inmetabolic cages for urine collection over the same period of time. Theurine samples were then stored at −80° C. until Mn analysis. Beforeanalysis, the samples were thawed and extensively shaken to obtainhomogenous samples. A five ml aliquot was taken from each sample and 5ml concentrated nitric acid was added. The samples were then resolved ina microwave oven and thereafter diluted with distilled water to a finalvolume of 50 ml. The Mn content of each sample was analyzed by ICP-MS(Inductively Coupled Plasma Mass Spectrometry). Identical samples ofcalmangafodipir and mangafodipir as those injected in the rats (i.e.,0.25 ml) were withdrawn and injected into test tubes. These samples weretreated in an identical manner to that of the urine samples and analyzedfor their Mn content. Results are presented as total 0-24 h urine Mncontent (expressed as μmol/kg±S.E.M.) and as percentage (±S.E.M.) of theinjected dose. The statistical difference between animals receivingcalmangafodipir and mangafodipir, with respect to excretion of manganeseinto the urine, was tested by an unpaired Student's t-test. A p-valuelower than 0.05 was considered as a statistically significantdifference.

Results

Results are set forth in FIGS. 6A, 6B and 6C. Twenty-four hours after ivinjection of 0.25 ml 10 mM mangafodipir containing 2.59 μmol manganese(Mn), 0.60±0.04 μmol Mn was recovered in urine (FIG. 6A), correspondingto 23.1±1.4% of the injected dose (after the basal excretion of 0.035μmol has been subtracted, FIG. 6B). The corresponding figure afterinjection of 0.25 ml 50 mM calmangafodipir containing 2.52 μmol Mn was1.27±0.07 μmol Mn (FIG. 6A), corresponding to 50.5±2.6% of the injecteddose (FIG. 6B). The difference between mangafodipir and calmangafodipirwas highly significant (p<0.0001). The difference in renal Mn excretionwas more or less reflected in the difference in renal excretion of zinc(Zn); expressed as increased Zn excretion, i.e., the basal 24 hexcretion (0.068 μmol) is subtracted (FIG. 6C).

Conclusion

Thus, at an equivalent Mn dose, calmangafodipir doubled Mn excretion inurine in comparison to mangafodipir. The percentage Mn excreted in urineduring 0-24 h after intravenous injection of mangafodipir correspondsvery well with previously reported figures in rats (Hustvedt et al.,1997) and humans (Toft et al., 1997). The present results demonstratethat calmangafodipir releases much less Mn under in vivo conditions thanmangafodipir. This provides significant advantages in that the amount offree Mn available for uptake by the brain and other organs is reducedand that the therapeutic index is significantly increased as more oftherapeutic mangafodipir or its dephosphorylated counterparts, MnDPMPand MnPLED, are available in vivo. Thus, calmangafodipir renders atherapeutic treatment considerably less toxic and much more efficaciousthan that of mangafodipir.

Example 5

This example compares the cytoprotective effect of calmangafodipir withthat of mangafodipir and MnPLED with respect to myelosuppressive effectsof oxaliplatin in balb/c mice.

Method

In a first series of experiments, 3 groups each consisting of 5 femalebalb/c mice were treated once intraperitoneally with oxaliplatin at 7.5,10.0 and 12.5 mg/kg oxaliplatin, respectively. One day before (baseline)as well as 3 and 6 days after oxaliplatin treatment 50 μl EDTA bloodsamples were taken from the orbital venous plexus with a glasscapillary. The blood samples were analyzed using the automated systemCELL-DYN® Emerald (Abbott Diagnostics) for the content of white bloodcells (WBC), lymphocytes (LYM), neutrophils (NEU) and platelets (PLC).From the results (FIGS. 7A-7D) it was concluded that further experimentstesting the myeloprotective effects of calmangafodipir, mangafodipir andMnPLED should be performed at 12.5 mg/kg oxaliplatin and that blood cellsample analyses should be performed the day before and 6 days afteroxaliplatin administration in every mice. Thirty minutes beforeadministration of oxaliplatin (12.5 mg/kg) and 24 hours after, the micereceived saline, calmangafodipir (5 mg/kg; lot #11AK0105B), mangafodipir(1 and 10 mg/kg; lot #02090106) or MnPLED (1 mg/kg), intravenously (5mice in each group). A dose of 5 mg/kg calmangafodipir contained thesame amount of manganese as that of 1 mg/kg mangafodipir, i.e., 1.3μmol; 1 mg/kg of MnPLED contained somewhat more manganese (approximately2 μmol). A control group received instead of oxaliplatin vehicle (5%glucose) and saline. The results are presented in graphs as relativechanges from baseline for the various treatments and blood cells(±S.E.M.). The statistical differences between treatment groups, whereappropriate, were tested by an unpaired Student's t-test. A p-valuelower than 0.05 was considered as a statistically significantdifference.

Results

The results are set forth in FIGS. 8A-8D. At an equivalent manganesedose, i.e., 5 mg/kg calmangafodipir was statistically significant moreefficacious than 1 mg/kg mangafodipir to protect the mice fromoxaliplatin-induced fall in total number of white blood cells (WBC)(FIG. 8A). A single dose of 12.5 mg/kg oxaliplatin caused the WBC tofall more than 80%, whereas the fall in animals treated withcalmangafodipir was only about 25%. The corresponding fall in micetreated with 1 or 10 mg/kg mangafodipir was around 50%. These resultspresumably also suggest that MnDPDP has to be dephosphorylated intoMnPLED before it can exert myeloprotective effects; 1 mg/kg MnPLED was,like calmangafodipir, significantly more efficacious than 1 and 10 mg/kgmangafodipir protecting WBC. Similar falls were seen in lymphocytes(LYM; FIG. 8B) and in neutrophils (NEU; FIG. 8C) after oxaliplatintreatment. Qualitatively similar results were also obtained whenneutrophils (NEU) were analyzed (FIG. 8C). Regarding platelets (PLC;FIGS. 7D and 8D, in comparison to WBC, LYM and NEU they differed both inthe sensitivity towards oxaliplatin and the cytoprotective effects ofthe test substances.

Conclusion

Calmangafodipir was at an equimolar manganese dose significantly morepotent than mangafodipir to protect balb/c mice against myelosuppressiveeffects of the anticancer drug oxaliplatin.

Example 6

The cytotoxic activity in murine colon cancer cells of calmangafodipirwas compared with that of mangafodipir, fodipir, MnPLED, ZnPLED, ZnDPDP,calfodipir (CaDPDP), PLED, and CaCl₂.

Method

The viability of cells was measured using the MTT assay. Briefly, 8,000CT26 (mouse colon carcinoma) cells were seeded per well on a 96-wellplate and grown over night in RPMI (Roswell Park Memorial Institute)1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100UI/ml penicillin and 100 μg/ml streptomycin at 37° C. in humidified airwith 5% CO₂. Cells were then exposed for 48 h to 1-1,000 μMcalmangafodipir (lot #11AK0105B), fodipir (DPDP; lot #RDL02090206), PLED(lot #KER-AO-122(2)), calfodipir (CaDPDP), mangafodipir (lot #02090106),MnPLED, ZnPLED, ZnDPMP and CaCl₂ at 37° C. The viability of the cellswas then assessed by adding 5 mg/ml methylthiazoletetrazolium (MTT) to afinal concentration of 0.5 mg/ml and incubating cells for a further 4 hat 37° C. The blue formazan that is formed by mitochondrialdehydrogenases of viable cells was then dissolved over night at 37° C.by adding 10% SDS and 10 mM HCl to a final concentration of 5% SDS and 5mM HCl. Finally, the absorbance of the solution was read at 570 nm witha reference at 670 nm in a microplate reader Spectramax 340 (MolecularDevices, Sunnyvale, Calif., USA) connected to an Apple Macintoshcomputer running the program Softmax Pro V1.2.0 (Molecular Devices,Sunnyvale, Calif., USA).

Results

The cytotoxic activity of fodipir, PLED, calfodipir or calmangafodipirwas about 20 times higher than that of mangafodipir (FIGS. 9A and 9B).MnPLED was almost 10 times more potent than mangafodipir in its abilityto kill CT26 cancer cells (FIG. 9A). Neither ZnDPDP, ZnPLED nor CaCl₂displayed any cytotoxic activity at all at the used concentrations(FIGS. 9A and 9B).

Conclusion

When calmangafodipir and mangafodipir were compared, calmangafodipir wasfound to be about 20 times more potent than mangafodipir to kill CT26cancer cells. Dissociation of manganese to some extent from fodipirprobably explains the cancer killing efficacy of mangafodipir.Calmangafodipir as defined in Examples 1 and 3, at manganese equimolarconcentrations, is on the other hand as efficacious as fodipir alone,i.e., the killing efficacy of calmangafodipir is much higher than thatof mangafodipir at equimolar manganese concentrations. This findingsuggests two important properties. Firstly, dephosphorylated PLED isprobably as efficacious as its phosphorylated counterpart fodipir withrespect to its cancer cell killing ability, and secondly, the lowerstability of MnPLED in comparison to that of mangafodipir (Rocklage etal., 1989) probably explains the higher efficacy of MnPLED. The lack ofany cytotoxic activity of ZnDPDP and ZnPLED is presumably due to the1000 times higher stability of these complexes in comparison to theirmanganese counterparts (Rocklage et al., 1989).

Example 7

This example compares the antitumor activity of oxaliplatin in a murinecolon cancer (CT26)-bearing mice model in the presence and absence ofcalmangafodipir.

Method

CT26 cells were grown in 75 cm² culture flasks in RPMI (Roswell ParkMemorial Institute) 1640 medium containing 10% fetal bovine serum, 2 mML-glutamine, 100 UI/ml penicillin and 100 μg/ml streptomycin at 37° C.in humidified air with 5% CO₂. When the cells reached ˜50% confluencythey were harvested by trypsinization. Briefly, cells were washed withphosphate based saline (PBS) (pH 7.3) and exposed to 0.05% Trypsin/0.53mM EDTA at 37° C. for ˜5 min. The trypsinization was stopped by addingRPMI1640 culture medium. Cells were counted and centrifuged at 200×g for5 min. Thereafter, they were washed in PBS, centrifuged again andresuspended in PBS at a concentration of 2×10⁶/350 μl for injection intomice. Balb/c female mice between 6 and 8 weeks of age were used, asdescribed by Laurent et al., 2005. Briefly, each mouse was injectedsubcutaneously in the back of the neck with 2×10⁶ of CT26 cells at day0. After 7 days (day 7) when the tumors were detectable, the tumor sizewas determined with a caliper and mice were grouped (5 in each group) sothat the sizes of the tumors were not statistically different by group.Oxaliplatin±calmangafodipir (lot #11AK0105B) was injected and one groupof mice received vehicle (0.9% saline+5% glucose) treatment alone. In afirst series of experiments, mice were injected i.v. with saline or 50mg/kg calmangafodipir 30 minutes prior to i.p. administration of 20mg/kg oxaliplatin (diluted in 5% glucose) or 5% glucose. These micereceived in addition saline or 50 mg/kg calmangafodipir 24 hours later(day 8). In another series of experiments, mice were injected i.v. withsaline or 5 mg/kg calmangafodipir 30 minutes 10 mg/kg oxaliplatin(diluted in 5% glucose) or 5% glucose, and saline or 5 mg/kgcalmangafodipir 24 hours later (day 8). The mice were killed on day 10and the tumors were excised and wet weights were determined. Thestatistical differences between treatment groups, where appropriate,were tested by an unpaired Student's t-test. A p-value lower than 0.05was considered as a statistically significant difference.

Results

The results are set forth in FIGS. 10A and 10B. In the first series ofexperiment the mice received 20 mg/kg oxaliplatin, which is close to thehighest tolerated dose. Single treatment with oxaliplatin resulted in astatistically significant and more than 50% reduction in tumor weight.Treatment with calmangafodipir (50 mg/kg) did not have any negativeinfluence on the antitumor effect of oxaliplatin at a high dose (FIG.10A). However, in a second series of experiments in which 10 mg/kgoxaliplatin was used, treatment with a relatively low dose ofcalmangafodipir (5 mg/kg) resulted in a statistically significant betterantitumor effect (FIG. 10B); the combined effect of 10 mg/kg oxaliplatinplus 5 mg/kg calmangafodipir was almost as efficacious as 20 mg/kgoxaliplatin alone.

Conclusion

Calmangafodipir did not interfere negatively with the antitumor activityof oxaliplatin, and, to the contrary, at a relatively low dose ofoxaliplatin (10 mg(kg), calmangafodipir actually increased the antitumorefficacy.

Example 8

This example compares levels of manganese after repeated intravenousinjections of calmangafodipir and mangafodipir (39 times over 33 weeks)in the rat brain, pancreas and liver.

Method

Wistar male and female rats were intravenously injected with either 0.9%NaCl, 72.0 μmol/kg mangafodipir (lot #02090106; corresponding to 72μmol/kg of manganese) or 374.4 μmol/kg calmangafodipir (lot #11AK0105B;corresponding to 72 μmol/kg of manganese) 3 times a week for 13 weeks(each treatment group consisted of 9 males+9 females). Each dose ofcalmangafodipir corresponded to about 36 times the assumed clinical dose(ACD). After the 13-week administration period, the rats were sacrificedand the brains and pancreas were dissected out and approximately 0.5 gsamples were stored frozen until Mn analysis. The Mn content of eachsample was analyzed by ICP-MS. Results are expressed as μg/g wetweight±S.E.M. Statistical difference between the mangafodipir group andthe calmangafodipir group, with respect to Mn content, was tested by apaired Student's t-test. A p-value lower than 0.05 was considered as astatistically significant difference.

Results

Results are set forth in FIGS. 11A-C. The Mn brain content inNaCl-treated control rats, mangafodipir-treated rats, andcalmangafodipir-treated rats was 0.38±0.01, 0.99±0.02 and 0.74±0.01 μg/gw.w., respectively. The corresponding Mn content in the pancreas was1.66±0.06, 5.54±0.45 and 3.35±0.19 μmol/kg, respectively. Although, theMn content of the liver was statistically significant elevated in themangafodipir group (FIG. 11C), the relative elevation was much less thanthose seen in the brain and pancreas.

Conclusion

Administration of a high accumulated dose of calmangafodipir into ratsresults in significantly less retention of manganese in the brain andpancreas compared to mangafodipir (the total dose in both casescorresponded to approximately 2800 μmol/kg of manganese). These resultsdemonstrate the improved toxicological profile of calmangafodipir incomparison to that of mangafodipir.

Example 9

This example shows the cytoprotective effect of calmangafodipir withrespect to myelosuppressive effects of carboplatin in balb/c mice.

Method

In a first series of experiments, 3 groups, each consisting of 5 femalebalb/c mice, were treated once intraperitoneally with carboplatin at 75,100 and 125 mg/kg carboplatin, respectively. One day before (baseline),as well as 3 and 6 days after, carboplatin treatment, 50 μl EDTA bloodsamples were taken from the orbital venous plexus with a glasscapillary. The blood samples were analyzed using the automated systemCELL-DYN® Emerald (Abbott Diagnostics) for the content of white bloodcells (WBC), lymphocytes (LYM), neutrophils (NEU) and platelets (PLC).From the results (FIGS. 12A-12D), it was concluded that furtherexperiments testing the myeloprotective effect of calmangafodipir shouldbe performed at 125 mg/kg carboplatin and that, in case of WBC, NEU andLYM, blood cell sample analyses should be performed the day before and 3days after carboplatin administration, and, in case of PLC, blood cellsample analysis should be performed the day before and 6 days aftercarboplatin administration. Thirty minutes before administration ofcarboplatin (125 mg/kg) and 24 hours after, the mice received saline orcalmangafodipir (1, 3, 10 or 30 mg/kg; lot #11AK0105B). A control groupreceived vehicle (saline) and saline instead of carboplatin. The resultsare presented in graphs as relative changes from baseline for thevarious treatments (mean±S.E.M.). The statistical differences betweentreatment groups, where appropriate, were tested by an unpairedStudent's t-test. A p-value lower than 0.05 was considered as astatistically significant difference.

Results

The results are set forth in FIGS. 13A-13D. Carboplatin (125 mg/kg)caused an approximately 50% decrease in WBC, as well as in NEU and LYM.Treatment with calmangafodipir at a dose of 3 mg/kg abolished thesedecreases. The dose-response of calmangafodipir displayed a bell shapedappearance in each case, in a similar way as previously described formangafodipir with respect to its cardioprotective effect againstdoxorubicin in CD mice (Kurz et al., Transl Oncol 2012; 5:252-259).

Regarding platelets (PLC, FIGS. 12D and 13D), in comparison to WBC, LYMand NEU, they differed in the sensitivity towards carboplatin.

Conclusion

Calmangafodpir profoundly protects balb/c mice against myelosuppressiveeffects of the anticancer drug carboplatin.

Example 10

This example compares the antitumor activity of oxaliplatin in coloncancer (CT26)-bearing immune competent balb/c mice and immune deficientnude balb/c mice (nu/nu) in the presence and absence of calmangafodipir.

Method

CT26 cells were grown in 75 cm² culture flasks in RPMI 1640 mediumcontaining 10% fetal bovine serum, 2 mM L-glutamine, 100 UI/mlpenicillin and 100 μg/ml streptomycin at 37° C. in humidified air with5% CO₂. When the cells reached ˜50% confluency they were harvested bytrypsinization. Briefly, cells were washed with PBS (pH 7.3) and exposedto 0.05% Trypsin/0.53 mM EDTA at 37° C. for ˜5 min. The trypsinizationwas stopped by adding RPMI 1640 culture medium. Cells were counted andcentrifuged at 200×g for 5 min. Thereafter, they were washed in PBS,centrifuged again and resuspended in PBS at a concentration of 2×10⁶/350μl for injection into mice. Immune competent balb/c female mice (balb/c)and immune incompetent nude female balb/c mice (blab/c nu/nu) between 6and 8 weeks of age were used, as described by Laurent et al., 2005.Briefly, each mouse was injected subcutaneously in the back of the neckwith 2×10⁶ of CT26 cells at day 0. After 7 days (day 7) when the tumorswere detectable, the tumor size was determined with a caliper and micewere grouped (5 in each group) so that the sizes of the tumors were notstatistically different by group. Groups of mice (5 in each group, asillustrated in FIG. 14) were injected i.v. with saline or 5 mg/kgcalmangafodipir (lot #11AK0105B) 30 minutes prior to i.p. administrationof 10 mg/kg oxaliplatin (diluted in 5% glucose) or 5% glucose. Micereceived in addition saline or 5 mg/kg calmangafodipir 24 hours later(day 8). The mice were sacrificed on day 10 and the tumors were excisedand wet weights were determined. The results are presented in a graphfor the various treatments (mean±S.E.M.). The statistical differencesbetween treatment groups, where appropriate, were tested by an unpairedStudent's t-test. A p-value lower than 0.05 was considered as astatistically significant difference.

Results

The results are set forth in FIG. 14. There was clear tendency that thetumors grew larger in the immune deficient balb/c mice than in immunecompetent balb/c nu/nu mice but this difference did not reachstatistical significance (p=0.0870). A single treatment with 10 mg/kgoxaliplatin resulted in statistically insignificant 20 to 30% reductionin tumor weights in immune competent and immune deficient balb/c mice.Treatment with 5 mg/kg calmangafodipir did not have any negativeinfluence on the antitumor effect of oxaliplatin in either the immunecompetent or in the immune incompetent mice. The mean tumor weight wasactually statistically significantly reduced in the immune competentmice treated with 5 mg/kg calmangafodipir compared to controls. However,no such reduction was seen in the immune deficient mice.

Conclusion

Calmangafodipir did not interfere negatively with the antitumor activityof oxaliplatin in either immune competent or immune deficient mice butit was only in immune competent mice that calmangafodipir actuallyincreased the antitumor efficacy.

Example 11

The cytotoxic activity of calmangafodipir toward human non-small celllung cancer (NSCLC) U1810 cells and murine non-small cell lung cancer(LLC1) was compared with that of mangafodipir.

Methods

The viability of cells was measured using the MTT assay. Briefly, 8,000human U1810 NSCLC or LLC1 NSCLC cells were seeded per well on a 96-wellplate and grown over night in RPMI (Roswell Park Memorial Institute)1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100UI/ml penicillin and 100 μg/ml streptomycin at 37° C. in humidified airwith 5% CO₂. Cells were then exposed for 48 h to 1-1,000 μMcalmangafodipir (lot #11AK0105B) or mangafodipir (lot #02090106). Theviability of the cells was then assessed by adding 5 mg/mlmethylthiazoletetrazolium (MTT) to a final concentration of 0.5 mg/mland incubating cells for a further 4 h at 37° C. The blue formazan thatis formed by mitochondrial dehydrogenases of viable cells was thendissolved over night at 37° C. by adding 10% SDS and 10 mM HCl to afinal concentration of 5% SDS and 5 mM HCl. Finally, the absorbance ofthe solution was read at 570 nm with a reference at 670 nm in amicroplate reader Spectramax 340 (Molecular Devices, Sunnyvale, Calif.,USA) connected to an Apple Macintosh computer running the programSoftmax Pro V1.2.0 (Molecular Devices, Sunnyvale, Calif., USA). Theviability of U1810 or LLC1 cells in the presence of increasingconcentrations of calmangafodipir or mangafodipir is presented asconcentration response curves (mean±S.D.). The individual curves werefitted to the sigmoidal variable slope response logistic equation(Graphpad Prism, version 5.02). From this analysis the concentrationscausing 50% inhibition (IC₅₀) of the test substances were calculated.

Results

The cytotoxic activity of calmangafodipir and mangafodipir toward NSCLCU1810 and LLC1 cells is shown in FIGS. 15A and 15B. The calculated IC₅₀ratio between mangafodipir and calmangafodipir (0.0006329/0.00002274)showed that calmangafodipir was about 28 times more potent thanmangafodipir to kill U1810 cells (FIG. 15A). Although calmangafodipirwas significantly more potent than mangafodipir to kill LLC1 cells,because of the ambiguous appearance of the mangafodipir curve (FIG. 15B)it was not meaningful to calculate an IC₅₀ ratio between mangafodipirand calmangafodipir.

Conclusions

The results demonstrate the superior efficacy of calmangafodipir incomparison to mangafodipir to kill the non-small cell lung cancer cells,U1810 and LLC1.

The examples and specific embodiments set forth herein are illustrativein nature only and are not to be taken as limiting the scope of theinvention defined by the following claims. Additional specificembodiments and advantages of the present invention will be apparentfrom the present disclosure and are within the scope of the claimedinvention.

What is claimed is:
 1. A method for treatment of oxidative stressassociated with a pathological condition in a patient, the methodcomprising administering to the patient a mixed metal complex of acompound of Formula I, or a salt thereof, in an amount effective toreduce the oxidative stress, wherein the mixed metals comprise calciumand manganese, and wherein the molar ratio of calcium to manganese is1-10:

wherein X represents CH, each R¹ independently represents hydrogen or—CH₂COOH; each R² independently represents ZYR⁶ wherein Z represents abond, CH₂ or CH₂O; Y represents a bond or an oxygen atom; R⁶ is ahydrogen atom, COOR⁸, alkyl, alkenyl, cycloalkyl, aryl or aralkyl group,optionally substituted by one or more groups selected from COOR⁸, CONR⁸₂, NR⁸ ₂, OR⁸, ═NR⁸, ═O, OP(O)(OR⁸)R⁷ and OSO₃M; R⁷ is hydroxy,optionally hydroxylated, optionally alkoxylated alkyl or aminoalkylgroup; R⁸ is a hydrogen atom or an optionally hydroxylated, optionallyalkoxylated alkyl group; M is a hydrogen atom or one equivalent of aphysiologically tolerable cation; provided that each ZYR⁶ includes a—CH₂O— linkage to the respective pyridine ring; R³ represents ethylene;and each R⁴ independently represents hydrogen or C₁₋₃ alkyl.
 2. A methodaccording to claim 1, wherein in the mixed metal complex, R⁶ is a mono-or poly(hydroxy or alkoxylated) alkyl group or of the formulaOP(O)(OR⁸)R⁷; and R⁷ is hydroxy, or an unsubstituted alkyl or aminoalkylgroup.
 3. A method according to claim 1, wherein the compound of FormulaI is N,N′-bis-(pyridoxal-5-phosphate)-ethylenediamine-N,N′-diacetic acid(DPDP) or N,N′-dipyridoxyl ethylenediamine-N,N′-diacetic acid (PLED), ora pharmaceutically acceptable salt thereof.
 4. A method according toclaim 3, wherein the mixed metal complex has a Ca²⁺/Mn²⁺ molar ratio ofabout
 4. 5. A method according to claim 3, wherein the mixed metalcomplex is a mixed metal complex of a sodium salt of a compound ofFormula I.
 6. A method according to claim 3, wherein the mixed metalcomplex is administered in a pharmaceutical composition comprising themixed metal complex and one or more physiologically acceptable carriersand/or excipients.
 7. A method according to claim 1, wherein the mixedmetal complex has a Ca²⁺/Mn²⁺ molar ratio of about
 4. 8. A methodaccording to claim 1, wherein the mixed metal complex is a mixed metalcomplex of a sodium salt of a compound of Formula I.
 9. A methodaccording to claim 1, wherein the mixed metal complex is freeze dried.10. A method according to claim 1, wherein the mixed metal complex isadministered in a pharmaceutical composition comprising the mixed metalcomplex and one or more physiologically acceptable carriers and/orexcipients.
 11. A method according to claim 1, comprising administeringabout 0.01 to 50 μmol/kg body weight of the mixed metal complex.
 12. Amethod according to claim 1, comprising administering about 0.1 to 10μmol/kg body weight of the mixed metal complex.
 13. A method accordingto claim 1, comprising administering about 0.1 to 5 μmol/kg body weightof the mixed metal complex.
 14. A method according to claim 1, whereinthe pathological condition is an ischemia-reperfusion-induced injury.15. A method according to claim 1, wherein the pathological condition isassociated with atherosclerosis.
 16. A method according to claim 1,wherein the pathological condition is associated with diabetes.
 17. Amethod according to claim 1, wherein the pathological condition isassociated with a thrombolytic treatment, a cardiopulmonary bypass, orpercutaneous transluminal angioplasty.
 18. A method according to claim1, wherein the pathological condition is a result of cardiac or organtransplantation surgery.
 19. A method according to claim 1, wherein thepathological condition is a result of stroke.
 20. A method according toclaim 1, wherein the pathological condition is a pathological conditionof iron or copper.
 21. A method according to claim 1, wherein thepathological condition is thalassemia, sickle cell anemia, transfusionalhemosiderosis, or Wilson's disease.
 22. A method according to claim 1,wherein the pathological condition is hepatitis-induced liver cirrhosis,non-alcoholic steatohepatitis (NASH), or viral-induced chronichepatitis.
 23. A method according to claim 1, wherein the pathologicalcondition is a radiation-induced injury.
 24. A method according to claim1, wherein the pathological condition is cancer.
 25. A method accordingto claim 1, wherein the pathological condition is non-small cell lungcancer, colorectal cancer, prostate cancer, breast cancer, pancreaticcancer, or malignant melanoma.
 26. A method of treatment of prostatecancer, breast cancer, pancreatic cancer, or malignant melanoma, themethod comprising administering to the patient an effective amount of amixed metal complex of a compound of Formula I, or a salt thereof,wherein the mixed metals comprise calcium and manganese and wherein themolar ratio of calcium to manganese is 1-10:

wherein X represents CH, each R¹ independently represents hydrogen or—CH₂COOH; each R² independently represents ZYR⁶ wherein Z represents abond, CH₂ or CH₂O; Y represents a bond or an oxygen atom; R⁶ is ahydrogen atom, COOR⁸, alkyl, alkenyl, cycloalkyl, aryl or aralkyl group,optionally substituted by one or more groups selected from COOR⁸, CONR⁸₂, NR⁸ ₂, OR⁸, ═NR⁸, ═O, OP(O)(OR⁸)R⁷ and OSO₃M; R⁷ is hydroxy,optionally hydroxylated, optionally alkoxylated alkyl or aminoalkylgroup; R⁸ is a hydrogen atom or an optionally hydroxylated, optionallyalkoxylated alkyl group; M is a hydrogen atom or one equivalent of aphysiologically tolerable cation; provided that each ZYR⁶ includes a—CH₂O— linkage to the respective pyridine ring; R³ represents ethylene;and each R⁴ independently represents hydrogen or C₁₋₃ alkyl.
 27. Amethod of treatment of a pathological condition in a patient, whereinthe pathological condition is ischemia-reperfusion-induced injury; iscaused by oxidative stress in a thrombolytic treatment, acardiopulmonary bypass, percutaneous transluminal angioplasty, and/oratherosclerosis; or is a result of oxidative stress in cardiac or organtransplantation surgery or stroke, the method comprising administeringto the patient an effective amount of a mixed metal complex of acompound of Formula I, or a salt thereof, wherein the mixed metalscomprise calcium and manganese and wherein the molar ratio of calcium tomanganese is 1-10:

wherein X represents CH, each R¹ independently represents hydrogen or—CH₂COOH; each R² independently represents ZYR⁶ wherein Z represents abond, CH₂ or CH₂O; Y represents a bond or an oxygen atom; R⁶ is ahydrogen atom, COOR⁸, alkyl, alkenyl, cycloalkyl, aryl or aralkyl group,optionally substituted by one or more groups selected from COOR⁸, CONR⁸₂, NR⁸ ₂, OR⁸, ═NR⁸, ═O, OP(O)(OR⁸)R⁷ and OSO₃M; R⁷ is hydroxy,optionally hydroxylated, optionally alkoxylated alkyl or aminoalkylgroup; R⁸ is a hydrogen atom or an optionally hydroxylated, optionallyalkoxylated alkyl group; M is a hydrogen atom or one equivalent of aphysiologically tolerable cation; provided that each ZYR⁶ includes a—CH₂O— linkage to the respective pyridine ring; R³ represents ethylene;and each R⁴ independently represents hydrogen or C₁₋₃ alkyl.
 28. Amethod of treatment of a pathological condition of iron or copper andcaused by oxidative stress, the method comprising administering to thepatient an effective amount of a mixed metal complex of a compound ofFormula I, or a salt thereof, wherein the mixed metals comprise calciumand manganese and wherein the molar ratio of calcium to manganese is1-10:

wherein X represents CH, each R¹ independently represents hydrogen or—CH₂COOH; each R² independently represents ZYR⁶ wherein Z represents abond, CH₂ or CH₂O; Y represents a bond or an oxygen atom; R⁶ is ahydrogen atom, COOR⁸, alkyl, alkenyl, cycloalkyl, aryl or aralkyl group,optionally substituted by one or more groups selected from COOR⁸, CONR⁸₂, NR⁸ ₂, OR⁸, ═NR⁸, ═O, OP(O)(OR⁸)R⁷ and OSO₃M; R⁷ is hydroxy,optionally hydroxylated, optionally alkoxylated alkyl or aminoalkylgroup; R⁸ is a hydrogen atom or an optionally hydroxylated, optionallyalkoxylated alkyl group; M is a hydrogen atom or one equivalent of aphysiologically tolerable cation; provided that each ZYR⁶ includes a—CH₂O— linkage to the respective pyridine ring; R³ represents ethylene;and each R⁴ independently represents hydrogen or C₁₋₃ alkyl.
 29. Amethod of treatment of thalassemia, sickle cell anemia, transfusionalhemosiderosis, or Wilson's disease, in a patient, the method comprisingadministering to the patient an effective amount of a mixed metalcomplex of a compound of Formula I, or a salt thereof, wherein the mixedmetals comprise calcium and manganese and wherein the molar ratio ofcalcium to manganese is 1-10:

wherein X represents CH, each R¹ independently represents hydrogen or—CH₂COOH; each R² independently represents ZYR⁶ wherein Z represents abond, CH₂ or CH₂O; Y represents a bond or an oxygen atom; R⁶ is ahydrogen atom, COOR⁸, alkyl, alkenyl, cycloalkyl, aryl or aralkyl group,optionally substituted by one or more groups selected from COOR⁸, CONR⁸₂, NR⁸ ₂, OR⁸, ═NR⁸, ═O, OP(O)(OR⁸)R⁷ and OSO₃M; R⁷ is hydroxy,optionally hydroxylated, optionally alkoxylated alkyl or aminoalkylgroup; R⁸ is a hydrogen atom or an optionally hydroxylated, optionallyalkoxylated alkyl group; M is a hydrogen atom or one equivalent of aphysiologically tolerable cation; provided that each ZYR⁶ includes a—CH₂O— linkage to the respective pyridine ring; R³ represents ethylene;and each R⁴ independently represents hydrogen or C₁₋₃ alkyl.
 30. Amethod of treatment of hepatitis-induced liver cirrhosis, non-alcoholicsteatohepatitis (NASH), or viral-induced chronic hepatitis, in apatient, the method comprising administering to the patient an effectiveamount of a mixed metal complex of a compound of Formula I, or a saltthereof, wherein the mixed metals comprise calcium and manganese andwherein the molar ratio of calcium to manganese is 1-10:

wherein X represents CH, each R¹ independently represents hydrogen or—CH₂COOH; each R² independently represents ZYR⁶ wherein Z represents abond, CH₂ or CH₂O; Y represents a bond or an oxygen atom; R⁶ is ahydrogen atom, COOR⁸, alkyl, alkenyl, cycloalkyl, aryl or aralkyl group,optionally substituted by one or more groups selected from COOR⁸, CONR⁸₂, NR⁸ ₂, OR⁸, ═NR⁸, ═O, OP(O)(OR⁸)R⁷ and OSO₃M; R⁷ is hydroxy,optionally hydroxylated, optionally alkoxylated alkyl or aminoalkylgroup; R⁸ is a hydrogen atom or an optionally hydroxylated, optionallyalkoxylated alkyl group; M is a hydrogen atom or one equivalent of aphysiologically tolerable cation; provided that each ZYR⁶ includes a—CH₂O— linkage to the respective pyridine ring; R³ represents; and eachR⁴ independently represents hydrogen or C₁₋₃ alkyl.
 31. A method oftreatment of radiation-induced injury in a patient, the methodcomprising administering to the patient an effective amount of a mixedmetal complex of a compound of Formula I, or a salt thereof, wherein themixed metals comprise calcium and manganese and wherein the molar ratioof calcium to manganese is 1-10:

wherein X represents CH, each R¹ independently represents hydrogen or—CH₂COOH; each R² independently represents ZYR⁶ wherein Z represents abond, CH₂ or CH₂O; Y represents a bond or an oxygen atom; R⁶ is ahydrogen atom, COOR⁸, alkyl, alkenyl, cycloalkyl, aryl or aralkyl group,optionally substituted by one or more groups selected from COOR⁸, CONR⁸₂, NR⁸ ₂, OR⁸, ═NR⁸, ═O, OP(O)(OR⁸)R⁷ and OSO₃M; R⁷ is hydroxy,optionally hydroxylated, optionally alkoxylated alkyl or aminoalkylgroup; R⁸ is a hydrogen atom or an optionally hydroxylated, optionallyalkoxylated alkyl group; M is a hydrogen atom or one equivalent of aphysiologically tolerable cation; provided that each ZYR⁶ includes a—CH₂O— linkage to the respective pyridine ring; R³ represents ethylene;and each R⁴ independently represents hydrogen or C₁₋₃ alkyl.
 32. Amethod of treatment of a pathological condition associated with diabetesin a patient, the method comprising administering to the patient aneffective amount of a mixed metal complex of a compound of Formula I, ora salt thereof, wherein the mixed metals comprise calcium and manganeseand wherein the molar ratio of calcium to manganese is 1-10:

wherein X represents CH, each R¹ independently represents hydrogen or—CH₂COOH; each R² independently represents ZYR⁶ wherein Z represents abond, CH₂ or CH₂O; Y represents a bond or an oxygen atom; R⁶ is ahydrogen atom, COOR⁸, alkyl, alkenyl, cycloalkyl, aryl or aralkyl group,optionally substituted by one or more groups selected from COOR⁸, CONR⁸₂, NR⁸ ₂, OR⁸, ═NR⁸, ═O, OP(O)(OR⁸)R⁷ and OSO₃M; R⁷ is hydroxy,optionally hydroxylated, optionally alkoxylated alkyl or aminoalkylgroup; R⁸ is a hydrogen atom or an optionally hydroxylated, optionallyalkoxylated alkyl group; M is a hydrogen atom or one equivalent of aphysiologically tolerable cation; provided that each ZYR⁶ includes a—CH₂O— linkage to the respective pyridine ring; R³ represents ethylene;and each R⁴ independently represents hydrogen or C₁₋₃ alkyl.
 33. Amethod according to claim 1, wherein each R² independently represents—CH₂—OP(O)(OR⁸)R⁷.
 34. A method according to claim 26, wherein each R²independently represents —CH₂—OP(O)(OR⁸)R⁷.
 35. A method according toclaim 27, wherein each R² independently represents —CH₂—OP(O)(OR⁸)R⁷.36. A method according to claim 28, wherein each R² independentlyrepresents —CH₂—OP(O)(OR⁸)R⁷.
 37. A method according to claim 29,wherein each R² independently represents —CH₂—OP(O)(OR⁸)R⁷.
 38. A methodaccording to claim 30, wherein each R² independently represents—CH₂—OP(O)(OR⁸)R⁷.
 39. A method according to claim 31, wherein each R²independently represents —CH₂—OP(O)(OR⁸)R⁷.
 40. A method according toclaim 32, wherein each R² independently represents —CH₂—OP(O)(OR⁸)R⁷.